40
`
`
`
`Nature Vol. 281
`
`6 September 1979
`
`tryptic peptides. A 1-ml
`Fig. 5 Fingerprints of
`aliquot of SVB”"“‘«3-infected
`cell
`extract was
`immunoprecipitated as described in Fig. 4 legend,
`eluted in SDS sample buffer, mixed with Sug of
`unlabelled mouse globin and electrophoresed through
`a 20% polyacrylamide-SDS gel. A sample of authen-
`tic ”S—methionine-labelled mouse 13”"-globin from
`induced Friend cells was electrophoresed in a separate
`track. The globin bands were identified by staining,
`excised, washed with 10% methanol, then digested in
`situ with trypsin—TPCK lworthington)“. The super-
`natants were lyophilised, resuspended in 25 ul of
`H20 and analysed by two-dimensional ascending
`chromatography on 20><20cm cellulose thin-layer
`plates (Brinkman). The first solvent was isoamyl
`alcohol/pyridine/H20 (9O:120:105)22. The second
`solvent was
`butanol/pyridine/ acetic
`acid/H20
`(32.5:2S:5:20) containing 7% (w/v) 2,5-diphenyl-
`oxazole“. The autoradiographs were exposed for 8 d
`at -70 "C. a, Globin from SV)9"“‘-3-infected cells; b,
`authentic mouse B'““-globin; c, mixture of globin
`from SVB"“’-infected cells and authentic mouse 3””
`globin (one-half the amounts used in a and b).
`
`can be inserted into the late region of the recombinant if it is to
`be encapsidated into virions. This obstacle might be overcome
`by propagating the hybrid genome as an episome in persistently
`infected monkey cells’, thus overcoming the requirement for
`encapsidation. Furthermore, it is clear that, although we have
`
`concentrated on the globin system, the approach should also be
`applicable to a variety of other genes.
`We thank Drs K. Smith and S. Boyer for anti-B -globin
`antibody, B. Norman for preparing probes, and Ms C. Harvey
`for assisting in the preparation of the manuscript.
`
`Received 23 April; accepted 6 August 1979.
`
`a~}I-t~'.-:tu:—-
`
`Tilghman. S. M.. et al. Proc. narn. Acad. Sci. U.S.A. 74. 4406-4410 (1977).
`Tilghman, S. etal. Proc. natn. Acad. Sci. USA. 75. 725-729 (1978).
`Tiemeier, D. C. et ul. Cell)-I, 237-245 (1978).
`L/eder, A. et al. Proc. natn. Acad. Sci. U.S.A. 75, 6187-6191 (1978).
`Konkel, D. A., Tilghman, S. M. & Leder, P. Cell 15, 1125-1132 (1978).
`Hamer, D. 11., Davoli. D., Thomas, C. A. Jr & Fareed. G. C. J. malec. Biol. 112. 155-182
`(1977).
`7. Hamer, D. H. in Recombinant Molecules: Impacran Science and Society (eds Beers, R. F. &
`Bassett, E. G.) 317-336 (Raven, New York, 1977).
`8. Hamer, D. H. in Genetic Manipulation as it Affect: the Cancer Problem, 37-47 (Academic,
`New York, 1977).
`9. Upcroft, P. et al. Pmc. natn. Acnd. Sci. U.S.A. 75, 2117-2121 (1978).
`10. Hamel, D. 14.. Smith. K. D., Boyer, S. H. 1:. Leder, P. Cell 17, 725-736 (1979).
`11. Mulligan, R., Howard, B. & Berg, P. Nature 277. 108-114 (1979).
`12. Hamer, D. H. & Leder, P. Cell 17, 737-747 (1979).
`13. Scherrer. K. in Fundamental Techniques in Virology (eds Habel. K. & Salzman, N. P.)
`413-432 (Academic, New York, 1969).
`14. Aviv, H. & Leder, P. Proc. natn. Acad. Sci. U.S.A. 69, 1408-1412 (1972).
`15. McMaster, G. K. & Carmichael, G. G. Proc. natn. Acad. Sci. U.S.A. 74, 4835-4838 (1977).
`
`16. Alwine, .1. C., Kemp, D. J. & Stark, G. R. Proc. natn. Acad. Sci. U.$.A. 74, 5350-5354
`(1977).
`17. Ghnsh, P. (1 al. J. bial. Chem. 253, 3643-3647 (1978).
`18. Lai, C. 1.. Dhar, R. & Khoury, G. Cell 14, 971-982 (1973).
`19. Maize]. J. V. Jr in Methods in Virology Vol. 5. 179-246 (Academic, New York, 1971).
`20. O'Farrell, 1’. Z., Goodman, H. M. & O‘Farre]l. P. H. Cell 12, 1133-1142 (1977).
`21. Elder, J. H., Pickett, R. A., Hampton, I. & Lerner, R. A. J’ bial. Chem. 252, 6510-6515
`(1977).
`22. Baglioni. C. Biochim. biophys. Acta 59, 437-440 (1962).
`23. Gilman, J. G. Science 178, 868-874 (1972).
`24. Wigler, M., Pellicer, A., Silverstein, S. & Axel, R. Cell 14, 725-731 (1978).
`25. Weissmann, C. etal. 11th Miami Winter Symp. 1979 (in the press).
`26. Bolivar, F. et al. Gene 2. 95-113 (1977).
`27. Fiers, W. 21 al. Nature 273, 113-120 (1978).
`28. Raddy, V. B. et al. Science 200, 494-502 (1978).
`29. Hirt, B. J. molec. Biol. 26, 365-369 (1967).
`30. Deisseroth, A., Barker, 1., Anderson, W. F. & Nienhuis, A. Proc. natn. Acad. Sci. U.S.A.
`72, 2682-2686 (1975).
`31. Clegg, J. 3., Naughton, M. A. & Weatherall, D. F. I. malec. Biol. 19, 91-108 (1966).
`32. Kessler. S. W. J. Immun. 115, 1617-1624 (1975).
`33. Bonner, W. M. & Laslteyt, R. A. Eur. J. Biochem. 46. 83-88 (1974).
`34. Goff, S. P. & Berg, P. Cell 9, 695-705 (1976).
`
`
`
`Rabbit B-globin mRNA production in
`mouse L cells transformed with
`cloned rabbit B-globin chromosomal DNA
`Ned Mantei, Werner Boll & Charles Weissmann
`Institut fiir Molekularbiologie I, Universitéit Ziirich, 8093 Ziirich, Switzerland
`
`
`Mouse thymidine kinase-negative L cells were transformed
`with a cloned rabbit chromosomal B-globin gene linked to
`the cloned thymidine kinase gene of herpes simplex virus
`type 1. Most thymidine kinase-positive cell lines contained
`one or more copies of rabbit B—globin DNA and produced
`up to 2,000 copies of rabbit B-globin RNA per cell
`indistinguishable from its authentic counterpart. No mouse
`B-globin mRNA was detected.
`
`
`RECOMBINANT DNA technology has made it possible to iso-
`late, amplify and determine the primary structure of any
`eukaryotic DNA segment of interest. To explore the functional
`
`0028-0836/79/360040-07501.00
`
`significance of particular regions of a genome, it is desirable to
`generate localised modifications in the nucleic acid and then
`evaluate the resulting changes in the biological properties of the
`genome, in vitro or in viva (see ref. 1). The reintroduction of
`defined nucleic acid segments, in particular of cloned DNA, into
`eukaryotic cells has been accomplished by various methods.
`Mechanical microinjection of RNA or DNA into oocytes“ or
`into somatic cells‘ delivers a large dose of nucleic acid to a
`relatively small number of identifiable cells. Biophysical and
`biochemical approaches use liposomes” or erythrocytes7
`loaded with RNA which are fused with the target cells, or direct
`treatment of target cells with nucleic acid in the presence of
`facilitating agents3’“. Eflicient delivery of DNA to a large
`population of cells is attained by the use of DNA incorporated
`into viral particles”.
`
`© Macmillan Journals 1979
`
`Merck Ex. 1038, pg 1137
`
`Merck Ex. 1038, pg 1137
`
`
`
`
`
`Nature Vol. 281
`
`6 September 1979
`
`tryptic peptides. A 1-ml
`Fig. 5 Fingerprints of
`aliquot of SVB”"“‘«3-infected
`cell
`extract was
`immunoprecipitated as described in Fig. 4 legend,
`eluted in SDS sample buffer, mixed with Sug of
`unlabelled mouse globin and electrophoresed through
`a 20% polyacrylamide-SDS gel. A sample of authen-
`tic ”S—methionine-labelled mouse 13”"-globin from
`induced Friend cells was electrophoresed in a separate
`track. The globin bands were identified by staining,
`excised, washed with 10% methanol, then digested in
`situ with trypsin—TPCK lworthington)“. The super-
`natants were lyophilised, resuspended in 25 ul of
`H20 and analysed by two-dimensional ascending
`chromatography on 20><20cm cellulose thin-layer
`plates (Brinkman). The first solvent was isoamyl
`alcohol/pyridine/H20 (9O:120:105)22. The second
`solvent was
`butanol/pyridine/ acetic
`acid/H20
`(32.5:2S:5:20) containing 7% (w/v) 2,5-diphenyl-
`oxazole“. The autoradiographs were exposed for 8 d
`at -70 "C. a, Globin from SV)9"“‘-3-infected cells; b,
`authentic mouse B'““-globin; c, mixture of globin
`from SVB"“’-infected cells and authentic mouse 3””
`globin (one-half the amounts used in a and b).
`
`can be inserted into the late region of the recombinant if it is to
`be encapsidated into virions. This obstacle might be overcome
`by propagating the hybrid genome as an episome in persistently
`infected monkey cells’, thus overcoming the requirement for
`encapsidation. Furthermore, it is clear that, although we have
`
`concentrated on the globin system, the approach should also be
`applicable to a variety of other genes.
`We thank Drs K. Smith and S. Boyer for anti-B -globin
`antibody, B. Norman for preparing probes, and Ms C. Harvey
`for assisting in the preparation of the manuscript.
`
`Received 23 April; accepted 6 August 1979.
`
`a~}I-t~'.-:tu:—-
`
`Tilghman. S. M.. et al. Proc. narn. Acad. Sci. U.S.A. 74. 4406-4410 (1977).
`Tilghman, S. etal. Proc. natn. Acad. Sci. USA. 75. 725-729 (1978).
`Tiemeier, D. C. et ul. Cell)-I, 237-245 (1978).
`L/eder, A. et al. Proc. natn. Acad. Sci. U.S.A. 75, 6187-6191 (1978).
`Konkel, D. A., Tilghman, S. M. & Leder, P. Cell 15, 1125-1132 (1978).
`Hamer, D. 11., Davoli. D., Thomas, C. A. Jr & Fareed. G. C. J. malec. Biol. 112. 155-182
`(1977).
`7. Hamer, D. H. in Recombinant Molecules: Impacran Science and Society (eds Beers, R. F. &
`Bassett, E. G.) 317-336 (Raven, New York, 1977).
`8. Hamer, D. H. in Genetic Manipulation as it Affect: the Cancer Problem, 37-47 (Academic,
`New York, 1977).
`9. Upcroft, P. et al. Pmc. natn. Acnd. Sci. U.S.A. 75, 2117-2121 (1978).
`10. Hamel, D. 14.. Smith. K. D., Boyer, S. H. 1:. Leder, P. Cell 17, 725-736 (1979).
`11. Mulligan, R., Howard, B. & Berg, P. Nature 277. 108-114 (1979).
`12. Hamer, D. H. & Leder, P. Cell 17, 737-747 (1979).
`13. Scherrer. K. in Fundamental Techniques in Virology (eds Habel. K. & Salzman, N. P.)
`413-432 (Academic, New York, 1969).
`14. Aviv, H. & Leder, P. Proc. natn. Acad. Sci. U.S.A. 69, 1408-1412 (1972).
`15. McMaster, G. K. & Carmichael, G. G. Proc. natn. Acad. Sci. U.S.A. 74, 4835-4838 (1977).
`
`16. Alwine, .1. C., Kemp, D. J. & Stark, G. R. Proc. natn. Acad. Sci. U.$.A. 74, 5350-5354
`(1977).
`17. Ghnsh, P. (1 al. J. bial. Chem. 253, 3643-3647 (1978).
`18. Lai, C. 1.. Dhar, R. & Khoury, G. Cell 14, 971-982 (1973).
`19. Maize]. J. V. Jr in Methods in Virology Vol. 5. 179-246 (Academic, New York, 1971).
`20. O'Farrell, 1’. Z., Goodman, H. M. & O‘Farre]l. P. H. Cell 12, 1133-1142 (1977).
`21. Elder, J. H., Pickett, R. A., Hampton, I. & Lerner, R. A. J’ bial. Chem. 252, 6510-6515
`(1977).
`22. Baglioni. C. Biochim. biophys. Acta 59, 437-440 (1962).
`23. Gilman, J. G. Science 178, 868-874 (1972).
`24. Wigler, M., Pellicer, A., Silverstein, S. & Axel, R. Cell 14, 725-731 (1978).
`25. Weissmann, C. etal. 11th Miami Winter Symp. 1979 (in the press).
`26. Bolivar, F. et al. Gene 2. 95-113 (1977).
`27. Fiers, W. 21 al. Nature 273, 113-120 (1978).
`28. Raddy, V. B. et al. Science 200, 494-502 (1978).
`29. Hirt, B. J. molec. Biol. 26, 365-369 (1967).
`30. Deisseroth, A., Barker, 1., Anderson, W. F. & Nienhuis, A. Proc. natn. Acad. Sci. U.S.A.
`72, 2682-2686 (1975).
`31. Clegg, J. 3., Naughton, M. A. & Weatherall, D. F. I. malec. Biol. 19, 91-108 (1966).
`32. Kessler. S. W. J. Immun. 115, 1617-1624 (1975).
`33. Bonner, W. M. & Laslteyt, R. A. Eur. J. Biochem. 46. 83-88 (1974).
`34. Goff, S. P. & Berg, P. Cell 9, 695-705 (1976).
`
`
`
`Rabbit B-globin mRNA production in
`mouse L cells transformed with
`cloned rabbit B-globin chromosomal DNA
`Ned Mantei, Werner Boll & Charles Weissmann
`Institut fiir Molekularbiologie I, Universitéit Ziirich, 8093 Ziirich, Switzerland
`
`
`Mouse thymidine kinase-negative L cells were transformed
`with a cloned rabbit chromosomal B-globin gene linked to
`the cloned thymidine kinase gene of herpes simplex virus
`type 1. Most thymidine kinase-positive cell lines contained
`one or more copies of rabbit B—globin DNA and produced
`up to 2,000 copies of rabbit B-globin RNA per cell
`indistinguishable from its authentic counterpart. No mouse
`B-globin mRNA was detected.
`
`
`RECOMBINANT DNA technology has made it possible to iso-
`late, amplify and determine the primary structure of any
`eukaryotic DNA segment of interest. To explore the functional
`
`0028-0836/79/360040-07501.00
`
`significance of particular regions of a genome, it is desirable to
`generate localised modifications in the nucleic acid and then
`evaluate the resulting changes in the biological properties of the
`genome, in vitro or in viva (see ref. 1). The reintroduction of
`defined nucleic acid segments, in particular of cloned DNA, into
`eukaryotic cells has been accomplished by various methods.
`Mechanical microinjection of RNA or DNA into oocytes“ or
`into somatic cells‘ delivers a large dose of nucleic acid to a
`relatively small number of identifiable cells. Biophysical and
`biochemical approaches use liposomes” or erythrocytes7
`loaded with RNA which are fused with the target cells, or direct
`treatment of target cells with nucleic acid in the presence of
`facilitating agents3’“. Eflicient delivery of DNA to a large
`population of cells is attained by the use of DNA incorporated
`into viral particles”.
`
`© Macmillan Journals 1979
`
`Merck Ex. 1038, pg 1137
`
`Merck Ex. 1038, pg 1137
`
`
`
`
`
`© Nature Publishing Group1979
`
`Merck Ex. 1038, pg 1138
`
`
`
`
`
`© Nature Publishing Group1979
`
`Merck Ex. 1038, pg 1139
`
`
`
`
`
`© Nature Publishing Group1979
`
`Merck Ex. 1038, pg 1140
`
`
`
`
`
`© Nature Publishing Group1979
`
`Merck Ex. 1038, pg 1141
`
`
`
`
`
`© Nature Publishing Group1979
`
`Merck Ex. 1038, pg 1142
`
`
`
`
`
`© Nature Publishing Group1979
`
`Merck Ex. 1038, pg 1143
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